Summary of Work: Studies of chemical disposition are designed to provide both applied knowledge of the fate of chemicals in the intact animal in support of toxicity tests conducted by the National Toxicology Program and basic knowledge of mechanisms of chemical toxicity. Each study is designed to address those physical and chemical properties unique to the compounds studied as well as to provide data which will permit structural characterization of the respective chemical class. Each study is also designed to address the impact of one or more factors such as dose, age, sex or route of exposure on the toxicity of the chemical(s) studied and the significance of this data to assessments of human health risks. Studies conducted during the current year have addressed the fate and mechanisms of toxicity of 2-methylimidazole (2-MI) a synthetic intermediate widely used in the synthesis of pharmaceuticals, agricultural chemicals, dyes and in rubber manufacturing and the metabolism of model compounds by transgenic animals. Studies of 2-MI indicate that it is rapidly absorbed from the gastrointestinal tracts of both rats and mice and rapidly excreted, primarily in urine. Metabolism is not extensive and 70% of the material excreted is parent compound. It does, however, induce increased levels of glucuronosyltransferase that account for the more rapid clearance of thyroid hormones. Studies of xenobiotic metabolism by transgenic strains of mice are designed to confirm the utility of these strains as models for detection of carcinogenic agents. Three strains of transgenic mice, TG.AC, P-53 knockout and COX2, are being examined for their capacity to metabolize three xenobiotics, benzene, ethoxyquin and methacrylonitrile. Studies of these compounds offer the opportunity to examine the primary metabolic pathways and include several P-450 reactions as well as the primary phase two pathways. Summary of Work: 2,4-D is a herbicide with a high incidence of use world-wide and is one of four peroxisome proliferators being studied by the NTP. While 2,4-D is a fairly weak peroxisome proliferator in rodents, it was included because of the potential for human exposure and chemicals with a range of potency for causing proliferation were desired. We were interested in the sex-dependent variations in the clearance of 2,4-D in rats. Our earlier studies indicated that differences in metabolism or biliary excretion did not appear to be an explanation for the differences. Toxicokinetic analysis revealed significant differences in elimination and exposure parameters consistent with a greater ability to clear 2,4-D by male rats relative to females. This suggests that at equivalent doses, female rats are exposed to higher concentrations of 2,4-D for a longer time than males. The relatively low toxicity of 2,4- D has long been associated with the rapid elimination by renal tubular transport. There are no reported studies comparing active anion transport of 2,4-D in males and females of any species. Studies on sex and species differences of renal transport of 2,4-D may offer an explanation for the toxicokinetic differences observed. The fact that there are significant sex and species differences in the toxic effects of 2,4-D and similar chemicals makes the extrapolation from animal models to humans difficult. Greater understanding of the kinetic and toxicologic differences are required to determine which species best predict the potential for adverse human health effects. Phenolphthalein (PHTH) is present in many over-the-counter laxative preparations. It was the subject of an NTP 2-year toxicity/carcinogenicity study. In that study evidence for increased tumor incidence in multiple organs was found in both sexes and both species. As part of the effort to provide more information about how the NTP study may apply to human risk, we have performed metabolism and disposition studies with 14C-PHTH in male and female F344 rats and B6C3F1 mice. Metabolism of PHTH in humans and rodents is similar in that most of the drug is conjugated and eliminated. Recent work has concentrated on the identification of hydroxy- PHTH by NMR and mass spectrometry and confirmation by synthesis. The overall results in this study do not support the hypotheses that PHTH-dependent tumorigenesis in rodents is dependent on metabolism to a non-free radical reactive intermediate, or results from accumulation of products in target tissues. The results are consistent with proposals by other investigators that PHTH tumor induction is through generation of free radicals and/or the estrogenic activity of the parent compound. Hydroxy-PHTH, a catechol, could also be oxidized to a semiquinone free radical and may provide an additional source of radical-induced toxicity/carcinogenicity.